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Feeding pasteurized milk to suckling calves is a popular practice used increasingly on dairy farms. Waste milk is frequently fed to calves because of its high nutritional value and economic benefits compared to milk replacement products. However, one of the disadvantages of feeding waste milk is the potential for exposure to a high number of bacterial contaminants, which may lead to serious illnesses or infections in calves. One of these contaminants is Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of Johne's disease (paratuberculosis). The transmission and distribution of paratuberculosis in dairy herds occurs mostly through the feeding newborn calves with contaminated colostrum or milk, because this age group is believed to be most susceptible to infection. To reduce the risk of transmission of pathogens, on-farm pasteurization of milk has become increasingly popular. In this study, we analyzed the efficacy of a new commercial high-temperature, short-time pasteurizer (73.5°C for 20 to 25 s) in terms of MAP inactivation under experimental on-farm conditions. The pasteurizer uses a newly developed steam-heating technique, allowing for the pasteurization of the transition milk without clumping. In 3 independent trials, we spiked fresh raw milk samples to a level of 107 or 104 viable MAP cells/mL before pasteurization. We examined the thermal inactivation and viability of MAP using culture and a D29 bacteriophage-based assay. To verify the identity and number of MAP cells, we also performed PCR assays. Pasteurization of the inoculated milk (107 and 104 MAP cells/mL) resulted in a remarkable reduction in viable MAP cells. The mean inactivation rate of MAP ranged from 0.82 to 2.65 log 10 plaque-forming units/mL, depending on the initial MAP amount inoculated and the addition of conservative agents to the pasteurized milk. Nevertheless, approximately 103 MAP cells/mL remained viable and could be transferred to calves after high-temperature, short-time pasteurization of milk. [ABSTRACT FROM AUTHOR]

A survey of retail purchased semi-skimmed pasteurised milk ( n  = 368) for Mycobacterium avium subspecies paratuberculosis (MAP) was conducted between May 2014 and June 2015 across the midlands of England using the Phage-PCR assay. Overall, 10.3% of the total samples collected contained viable MAP cells, confirming that pasteurisation is not capable of fully eliminating human exposure to viable MAP through milk. Comparison of the results gained using the Phage-PCR assay with the results of surveys using either culture or direct PCR suggest that the phage-PCR assay is able to detect lower numbers of cells, resulting in an increase in the number of MAP-positive samples detected. Comparison of viable count and levels of MAP detected in bulk milk samples suggest that MAP is not primarily introduced into the milk by faecal contamination but rather are shed directly into the milk within the udder. In addition results detected an asymmetric distribution of MAP exists in the milk matrix prior to somatic cell lysis, indicating that the bacterial cells in naturally contaminated milk are clustered together and may primarily be located within somatic cells. These latter two results lead to the hypothesis that intracellular MAP within the somatic cells may be protected against heat inactivation during pasteurisation, accounting for the presence of low levels of MAP detected in retail milk. [ABSTRACT FROM AUTHOR]

Here an integrated three-step microbiological Fr 13 risk assessment (Zou and Davey, 2016) is synthesised for the first time. It is applied to vulnerability to failure of a batch-continuous pasteurizer for raw milk containing viable Mycobacterium avium subsp. paratuberculosis ( MAP ) - a chronic enteric pathogen linked to Crohn’s disease in humans. Pasteurization of raw milk is therefore globally important. The aim was to advance the Fr 13 risk framework to an integrated 3-step microbiological failure synthesis to study how naturally occurring fluctuations in processing can be transmitted and impact multi-step processes. The 3-steps of the pasteurizer are the heat-up of raw milk, holding for thermal inactivation of contaminant MAP , and; cool-down of the heat-treated milk. Overall pasteurizer failure is defined as unwanted survival of MAP following holding and is characterised by a risk factor ( p 2 ) for milk leaving this step. Realistic large-scale pasteurization is simulated using a refined Monte Carlo (with Latin Hypercube) sampling. Results show that for a (Regulatory) design reduction of log 10 = 5.5 (>99.999%) in viable MAP on a heat-up to 72 °C with 15 s holding, and a design safety margin of 2%, p 2 = 5.75%. This equates to up to ∼21 failures with unwanted MAP survival each year - averaged over an extended period of daily batch-continuous operations. We show these microbiological failures can be mitigated by installing precise safety-integrity-level (SIL) mass flow control on the input raw milk and by ensuring a holding time of ≥15 s. Results appear generalizable and therefore could be applied to a range of integrated 3-step microbiological processes. Findings will be of direct interest to risk analysts and milk processors and manufactures of equipment. The work is part of an investigation and development of Fr 13 as a new risk assessment and process design tool. [ABSTRACT FROM AUTHOR]

This Technical Research communication describes results of a study aimed at detecting the presence of Map in milk fed to calves, and identifying possible risk factors for that presence. A questionnaire was performed on 37 dairy farms and waste milk samples were collected on 3 occasions separated by a minimum of 1 week. For farms not feeding waste milk, bulk tank milk samples were collected instead. A real time PCR for the detection of the IS900 sequence was performed for the detection of Map. A majority of farms (89·2%) fed waste milk, with only one pasteurising the milk before feeding it to calves. Results of the PCR showed that 51·5% of the farms that were feeding waste milk had a positive result for Map on that milk. None of the studied risk factors were significantly associated with the presence of Map in milk samples, possibly due to the small number of farms entering the study. However, the prevalence of positive samples for Map on PCR was 3·5 times higher for farms that bought in animals from a single origin and 1·9 times higher for farms that bought from multiple farms, when compared with closed farms. Having a calving area for multiple cows also increased the risk of a positive Map result by 1·5 when compared with single pens. The risk of having a positive Map result on waste milk was 1·6 times higher for farms feeding that milk to male calves and 1·4 for farms feeding to both male and female calves, when compared with farms not feeding waste milk. This study highlights paratuberculosis as one of the potential risks of feeding waste milk to calves, and the need for mitigation strategies to be in place to avoid unnecessary disease transmission. [ABSTRACT FROM PUBLISHER]

In this study, twenty five bulk tank milk samples each of 50ml from Bulk tank milk of 25 holstein-friesian dairy herds representing 7 Egyptian Governorates, were collected at the period from January to July 2013. Each raw bulk tank milk sample was subjected to ELISA and real time PCR, while detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) was done by culture of milk samples on Herrold's egg yolk media "HEYM". The results indicated that real time PCR detected 17out of 25 samples (68 %) followed by ELISA 10 samples out of 25 (40%), more over the viable MAP was isolated from 15 out of 25 samples (60%), the autors indicated the high percentage of MAP isolation may be to milk contamination with infected faeces and shedded microorganisms directly in milk. The authors concluded that, Since the milk is considered an essential nutrient for childrens and adults, it should be free of any hazard pathogens could be transmitted to it by direct or indirect way, so it is very important in milk value chain to test the raw bulk milk provided to plant milk processing by efficient and fast test as real time PCR to exclude any possible threat for the consumer by using the efficient pasteurization for that pathogen. [ABSTRACT FROM AUTHOR]

Surveys from different parts of the world have reported that viable Mycobacterium avium subsp. paratuberculosis (MAP) can be cultured from approximately 2% of samples of retail pasteurised milk samples. Pasteurised milk is used for the production of powdered infant formula (PIF) and therefore there is a concern that MAP may also be present in these products. Several studies have previously reported the detection of MAP in PIF using PCR-based assays. However, culture-based surveys of PIF have not detected viable MAP. Here we describe a phage amplification assay coupled with PCR (page-PCR) that can rapidly detect viable MAP in PIF. The results of a small survey showed that the phage-PCR assay detected viable MAP in 13% (4/32) of PIF samples. Culture detected viable MAP in 9% (3/32) PIF samples, all of which were also phage-PCR positive. Direct IS 900 PCR detected MAP DNA in 22% (7/32) of PIF samples. The presence of viable MAP in PIF indicates that MAP either survived PIF manufacturing or that post-production contamination occurred. Irrespective of the route of MAP contamination, the presence of viable MAP in PIF is a potential public health concern. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the viability of Mycobacterium avium subsp. paratuberculosis (MAP), Escherichia coli (E. coli), and Salmonella Enteritidis (S. Enteritidis) during preparation and refrigerated storage of yogurt. Three yogurts were prepared using pasteurized commercial milk. Each yogurt was artificially contaminated with (1) MAP, (2) E. coli + S. Enteritidis, and (3) MAP + E. coli + S. Enteritidis. Samples were taken during and after the fermentation process until day 20 after inoculation. MAP was not detected during their preparation and short-term storage but was recuperated after starting at 180 min after inoculation storage. Live bacterial counts of E. coli, and S. Enteritidis increased during the first 24 hours, followed by a slight decrease towards the end of the study. In this study it was shown how MAP, E. coli, and S. Enteritidis resisted the acidic conditions generated during the preparation of yogurt and low storage temperatures. This work contributes to current knowledge regarding survival of MAP, E. coli, and S. Enteritidis during preparation and refrigerated storage of yogurt and emphasizes the need to improve hygiene measures to ensure the absence of these pathogenic microorganisms in dairy products. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 106 MAP cells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold''s egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP. [Copyright &y& Elsevier]

Consumer demand for food safety and quality improvements, combined with new regulations, requires determining the processor's confidence level that processes lowering safety risks while retaining quality will meet consumer expectations and regulatory requirements. Monte Carlo calculation procedures incorporate input data variability to obtain the statistical distribution of the output of prediction models. This advantage was used to analyze the survival risk of Mycobacterium avium subspecies paratuberculosis ( M. paratuberculosis) and Clostridium botulinum spores in high-temperature short-time (HTST) milk and canned mushrooms, respectively. The results showed an estimated 68.4% probability that the 15 sec HTST process would not achieve at least 5 decimal reductions in M. paratuberculosis counts. Although estimates of the raw milk load of this pathogen are not available to estimate the probability of finding it in pasteurized milk, the wide range of the estimated decimal reductions, reflecting the variability of the experimental data available, should be a concern to dairy processors. Knowledge of the C. botulinum initial load and decimal thermal time variability was used to estimate an 8.5 min thermal process time at 110 °C for canned mushrooms reducing the risk to 10−9 spores/container with a 95% confidence. This value was substantially higher than the one estimated using average values (6.0 min) with an unacceptable 68.6% probability of missing the desired processing objective. Finally, the benefit of reducing the variability in initial load and decimal thermal time was confirmed, achieving a 26.3% reduction in processing time when standard deviation values were lowered by 90%. Practical Application: In spite of novel technologies, commercialized or under development, thermal processing continues to be the most reliable and cost-effective alternative to deliver safe foods. However, the severity of the process should be assessed to avoid under- and over-processing and determine opportunities for improvement. This should include a systematic approach to consider variability in the parameters for the models used by food process engineers when designing a thermal process. The Monte Carlo procedure here presented is a tool to facilitate this task for the determination of process time at a constant lethal temperature. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium avium subspecies paratuberculosis (MAP) inactivation kinetics was studied to assess whether current legislative pasteurization prescriptions are sufficient to reduce this bacterium to an acceptable level in raw milk. To assess possible protective effects of milk components during pasteurization, raw milk and phosphate-buffered saline (PBS) were compared. Analyzing data from three replicate experiments in milk separately gave D60°C-values ranging from 114.3 to 244.5s and z-values ranging from 4.2 to 6.8°C; in PBS the ranges were 162.4 to 353.3s and 4.0 to 9.0°C, respectively. No statistically significant difference was observed between the heat resistance of MAP in milk versus PBS. The currently prescribed minimum HTST pasteurization conditions (71.7°C, 15s) were found to be insufficient to kill MAP in milk in 2 out of 6 replicate experiments, while LTLT pasteurization (minimum 62.7°C, 30min) was effective, based on extrapolation of the inactivation data obtained. [Copyright &y& Elsevier]

The objective was to evaluate if a standardized Johne's disease control program significantly reduced the prevalence of cattle infected with Mycobacterium avium ssp. paratuberculosis in dairy herds with a moderate to high initial infection prevalence of =10% ELISApositive adult cattle. Nine Wisconsin dairy herds of diverse sizes and management styles completed the 6-yr study. The control program involved changes to heifer rearing practices in combination with a routine testing program. For heifers, the program specifically required 1) segregated maternity pens for ELISA-positive and ELISA-negative cattle; 2) removal of calves from the maternity pen in <2 h; 3) use of colostrum only from individual ELISA-negative cows (no colostrum pooling); 4) hygienic collection of colostrum; 5) feeding of pasteurized milk as milk replacer or on-farm pasteurized milk until weaning; and 6) minimizing contact with manure from the adult cattle until weaning. The testing program was designed to detect the most infectious cattle by using a commercial ELISA once on every adult during each lactation. Producers were required to cull cows with strong-positive ELISA results before the next calving and to label cows with low- to medium-level ELISA results and manage them to limit infection transmission. Outcomes were measured by comparing the apparent prevalence based on ELISA or fecal culture in the whole herd and in first-lactation cohorts at 2 time points: before implementation of the control program and at the end of the trial. The combined results from the 9 herds showed a significant reduction in ELISA-positive cows, from 11.6% at the start of the trial to 5.6% at conclusion of the trial. The apparent prevalence decline among first-lactation cows was greater and was evident by ELISA (10.4 vs. 3.0%) and by fecal culture (17.0 vs. 9.5%). Although variations among farms were observed, the collective results demonstrated that bovine paratuberculosis can be controlled in dairy herds through effective heifer husbandry practices in combination with diagnostic testing to identify, for culling or management, cows most likely infectious. [ABSTRACT FROM AUTHOR]

Aims: To determine the effect of UV radiation on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map) inoculated into milk. Methods and Results: Mycobacterium avium ssp. paratuberculosis in a ultra heat treated milk matrix was subjected to increasing doses of UV-C radiation from 0 to 1836 mJ ml−1 using a pilot-scale UV reactor (20 l capacity). Survival of Map was monitored by culture on Herrold’s egg yolk medium, Middlebrook 7H10 medium and the FASTPlaqueTB™ phage assay. Differences in sensitivity to UV treatment were observed between strains, however, at 1000 mJ ml−1 a Map kill rate of 0·1–0·6 log10 was achieved regardless of strain used or method employed to enumerate Map. Although the inactivation trend was similar on the culture and phage assay, the former gave a consistently higher viable count. Conclusions: The use of UV radiation alone does not represent an alternative to current pasteurization regimes for a large reduction in viable Map in milk. Significance and Impact of the Study: To the authors’ knowledge the work here represents the first pilot-scale UV treatment process used to assess UV efficacy to inactivate Map in milk. The results are similar to those obtained with a laboratory-scale process indicating the difficulties associated with UV treatment of an opaque liquid and the recalcitrance of Map towards inimical treatments. [ABSTRACT FROM AUTHOR]

In the present study, the potential benefits of feeding pasteurized colostrum were demonstrated in calves born to dams naturally infected with Mycobacterium avium ssp. paratuberculosis. Calves were separated at birth from their dams and randomly allocated into a group fed either the colostrum of their dam (DC; n = 6), followed by feeding the milk of the dam for 3 wk and then milk replacer, or into a group fed pooled pasteurized colostrum (PC; n = 5) from healthy noninfected dams, followed by milk replacer. At 6 wk of age, calves were weaned onto calf starter, housed together, and fed in a similar manner throughout the rest of the 12-mo study. Calves were necropsied at the end of the study, and 25 tissue sites were sampled from each animal and cultured for M. avium ssp. paratuberculosis. Sixteen of the 25 tissue sites were positive for calves across both treatment groups, with 14 of the 16 tissue sites positive for DC calves and 9 of the 16 tissue sites positive for PC calves. The degree of colonization within a tissue was low and variable for calves within treatment groups, and fecal shedding of M. avium ssp. paratuberculosis was minimal during the 12-mo study. As a measure of the early immune response to infection, blood obtained from calves was stimulated in vitro with M. avium ssp. paratuberculosis antigen preparations, and IFN-y secretion was measured. Antigen-specific IFN-y was consistently greater throughout the study in DC calves (0.95 ± 0.19) compared with PC calves (0.43 ± 0.10). Although long-term benefits are unknown, these results indicate that feeding a source of colostrum from paratuberculosis-free dams may decrease the initial exposure of neonates to M. avium ssp. paratuberculosis, perhaps decreasing dissemination of infection over time. [ABSTRACT FROM AUTHOR]

Aims: To determine the effect of high pressures alone and in conjunction with pasteurization on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map). Methods and Results: Map in a milk matrix was subjected to 400, 500 and 600 MPa with and without pasteurization (72°C for 15 s) and plated onto Herrold’s egg yolk medium (HEYM) and Middlebrook 7H10 (7H10) agar, both containing antibiotic supplements. Medium 7H10 was found to give a significantly ( P < 0·001) better recovery than HEYM. A significantly greater ( P < 0·001) reduction in viable numbers was observed using 500 MPa (mean log reduction of 6·52) compared with 400 MPa (mean log reduction of 2·56) and between 400 MPa and control (no applied pressure) for 10 min treatments. A treatment time of 10 min resulted in significantly ( P < 0·001) fewer survivors than 5 min. Low numbers of survivors were still detected when pressure treatment at 400 and 600 MPa was combined with subsequent pasteurization. Conclusions: The use of high-pressure was effective in reducing viable numbers of Map but even when combined with pasteurization there were still survivors, albeit when high inoculum levels of Map were used. Significance and Impact of the Study: To the authors’ knowledge the work reported here represents the first study of the efficacy of high-pressure treatments alone and in combination with pasteurization to kill Map. The results indicate that further research is warranted before more commercial-scale studies are commissioned. [ABSTRACT FROM AUTHOR]

The effectiveness of high-temperature, short holding time (HTST) pasteurization and homogenization with respect to inactivation of Mycobacterium avium subsp. paratuberculosis was evaluated quantitatively. This allowed a detailed determination of inactivation kinetics. High concentrations of feces from cows with clinical symptoms of Johne's disease were used to contaminate raw milk in order to realistically mimic possible incidents most closely. Final M. avium subs p.paratuberculosis concentrations varying from 102 to 3.5 x 105 cells per ml raw milk were used. Heat treatments including industrial HTST were simulated on a pilot scale with 22 different time-temperature combinations, including 60 to 90°C at holding (mean residence) times of 6 to 15 s. Following 72°C and a holding time of 6 s, 70°C for 10 and 15 s, or under more stringent conditions, no viable M. avium subsp. paratuberculosis cells were recovered, resulting in >4.2- to >7.1-fold reductions, depending on the original inoculum concentrations. Inactivation kinetic modeling of 69 quantitative data points yielded an Eα of 305,635 J/mol and an Ink0 of 107.2, corresponding to a D value of 1.2 s at 72°C and a Z value of 7.7°C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at ≥72°C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis. [ABSTRACT FROM AUTHOR]

Raw cow's milk inoculated with four laboratory strains (102−105 cfu/mL) of Mycobacterium avium ssp. paratuberculosis (Map) was pasteurized in a custom designed pilot plant pasteurizer having a maximum throughput of 580 L/h under turbulent flow conditions. Following 16 pasteurizer trials none of the Map strains survived high-temperature short-time conditions (72.5°C × 27 s) whether milk was homogenized or not. Two dairy herds containing animals which were faecal positive for Map were sourced and milk was collected for pasteurization studies. Milk collected from one herd on five occasions in the autumn did not contain any detectable Map organisms, and the second herd that was sampled on only one occasion in early winter was shown to contain Map at low concentration. Map was not detected in any of these milks following pasteurization at 72.5°C for 27 s. Two natural isolates of Map inoculated into milk were likewise inactivated on pasteurization. [ABSTRACT FROM AUTHOR]

Abstract: Research has been focused on the detection of Mycobacterium avium subspecies paratuberculosis (MAP) in pasteurized milk; however, pasteurized milk is a key ingredient in a variety of food products. Therefore, MAP contamination in milk-derived products must be investigated. We undertook a six-month study to investigate the presence of viable MAP and MAP genetic components in cheese curds purchased from retail outlets in the northern and southern regions of Wisconsin and Minnesota. A total of 98 retail cheese curd samples were tested for MAP by PCR prescreen, culture on Herrold''s egg yolk agar slants with mycobactin J and amphoteracin B, naladixic acid, and vancomycin, and slant rinse PCR using IS900 and hspX primer sets. Although no viable MAP were able to be cultured, 5% of the samples were PCR positive with both the IS900 and hspX primer sets (MAP-specific DNA) when prescreened and 1% of the samples were PCR positive with both the IS900 and hspX primer sets when culture slants were rinsed and tested. [Copyright &y& Elsevier]

The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 10¹ to l05 M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in², applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or "miniclump" status by homogenization at 2,500 lb/in². Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization. [ABSTRACT FROM AUTHOR]

Cattle with Johne's disease can shed live Mycobacterium avium subsp. paratuberculosis (MAP) in their milk, and MAP can survive under simulated commercial pasteurization conditions. In several studies conducted in the United Kingdom and Canada, MAP DNA has been detected in retail pasteurized milk samples; however, in one study in the United Kingdom viable MAP was identified in commercially pasteurized milk. A double-blind study involving two laboratories was undertaken to evaluate retail pasteurized whole milk in the United States. Marshfield Clinic Laboratories used solid culture medium (Herrold's egg yolk agar slants with mycobactin J and amphotericin B, nalidixic acid, and vancomycin), and TREK Diagnostic Systems, Research and Development used liquid culture medium (ESP culture system). Cultures at both laboratories were confirmed by PCR. A total of 702 pints of retail whole milk were purchased in three of the top five milk-producing states (233 from California, 234 from Minnesota, and 235 from Wisconsin) over a 12-month period and were tested for the presence of viable MAP. The criteria used for identifying samples as positive for viable MAP were similar to those followed by most laboratories (positive culture with PCR confirmation). The combined data from the two laboratories revealed the presence of viable MAP in 2.8% of the retail whole milk pints tested. Although the number of samples containing viable MAP was similar among states (P > 0.05), there was a seasonal effect on the presence of viable MAP in retail milk (P = 0.05). More MAP-positive samples were identified during the third quarter of the year (July through September). Of the 22 brands of retail milk tested, 12 (55%) yielded at least one sample positive for viable MAP. [ABSTRACT FROM AUTHOR]

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp, paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp, paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail mill M. avium subsp, paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp, paratuberculosis. These findings confirm that M. avium subsp, paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp, paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route. [ABSTRACT FROM AUTHOR]

Mycobacterium avium subsp. paratuberculosis, the causative agent of a chronic enteritis in ruminants (Johne's disease), has been linked to Crohn's disease in humans. This microorganism is shed by infected animals primarily in the feces but is also shed in the milk at much lower levels. Therefore, dairy products from infected animals may be one mode of transmission of this animal pathogen. This study was designed to evaluate the effectiveness of the holder and high-temperature short-time pasteurization standards on the destruction of M. paratuberculosis. One hundred eighty experiments were conducted in this study using a slug-flow pasteurizer unit and a laboratory scale pasteurizer unit. Ultrahigh-temperature milk was inoculated at two concentrations, 108 and 105 CFU/ml, with three different field strains of M. paratuberculosis. Five different time-temperature combinations were evaluated: 62.7°C for 30 mm, 65.5°C for 16 s, 71.7°C for 15 s, 71.7°C for 20 s, and 74.4°C for 15 s. Three replicates of each experiment were run for the pasteurizer unit, time-temperature combination, and strain of M. paratuberculosis. Treatment of milk regardless of bacterial strain or pasteurizer unit resulted in an average 5.0- and 7.7-log kill for the low and high concentrations of inoculum, respectively. Milk treated for cheese production (65.5°C for 16 s) resulted in a much lower and more variable kill. Results from this study indicate that the current U.S. minimum standards for batch and high-temperature short-time pasteurization of grade A milk significantly reduced the survivability of M. paratuberculosis, but some bacteria survived subpasteurization heat treatment of milk used for cheese manufacture. [ABSTRACT FROM AUTHOR]

i.r. grant and m.t. rowe. 2004. To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R ( ca 106 CFU ml−1) were heated at 63°C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0·5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4°C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0·75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4°C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37°C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating ( P < 0·05) and after refrigerated storage for 48 h ( P < 0·01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates. CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample. Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies. [ABSTRACT FROM AUTHOR]

Johne's disease (JD) is a life-threatening gastrointestinal disease affecting ruminants, which causes crucial economical losses globally. This ailment is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious intracellular pathogen that belongs to the Mycobacteriaceae family. This acid-fast, hard-to-detect bacterium can resist milk pasteurization and be conveyed to dairy product consumers. Many studies have emphasized the zoonotic nature of MAP, suggesting an association between MAP and some gastroenteric conditions such as Crohn's disease in humans. This underlines the importance of utilizing efficient pasteurization alongside a state-of-the-art diagnostic system in order to minimize the possible ways this pathogen can be conveyed to humans. Until now, no confirmatory MAP screening technique has been developed that can reveal the stages of JD in infected animals. This is partially due to the lack of an efficient gold-standard reference method that can properly evaluate the performance of diagnostic assays. Therefore, the following research aimed to compare the merits of qPCR and ELISA assessments of milk for the detection of MAP in a total of 201 Sardinian unpasteurized sheep milk samples including 73 bulk tank milk (BTM) and 128 individual samples from a MAP-infected flock (MIF) applying various reference models. Accordingly, milk qPCR and ELISA assessments, together and individually, were used as reference models in the herd-level study, while serum ELISA and fecal PCR were similarly (together and in isolation) considered as the gold standards in the individual-level diagnosis. This study showed that the type of gold-standard test affects the sensitivity and specificity of milk qPCR and ELISA significantly. At the individual level in the MAP-infected flock, serum ELISA in isolation and together with fecal PCR were recognized as the best references; however, the best correlation was seen between milk and serum ELISA (p < 0.0001). Regarding the detection of MAP in BTM, qPCR IS900 was recognized as the most sensitive and specific diagnostic test (p < 0.0001) for monitoring the MAP shedders and animals with clinically developed symptoms within herds, under the condition that both milk qPCR and milk ELISA tests formed a binary reference model. The BTM analyses (qPCR and ELISA) revealed that MAP positivity has a seasonal pattern. This hypothesis was proven through a longitudinal study on 14 sheep herds. [ABSTRACT FROM AUTHOR]